Cardioprotective actions of estrogen in the prevention of heart disease in women have been well established. Effects of estrogens in men have been less widely studied. While it is likely that estrogen influences several different processes in the development of cardiovascular disease, it is conceivable that its mechanisms of action are gender-specific. Thus, it is important to understand the molecular and cellular basis for the differential actions of estrogens in men and women. The objective of this proposal is to examine the influence of estrogen on the expression of inducible nitric oxide synthase (iNOS), a gene that may be involved in the initiation of atherosclerosis in vascular smooth muscle (VSM). We have identified three possible mechanisms by which sex-dependent estrogen responsiveness may be mediated. Sex differences in the estrogen- enhanced induction of iNOS by IL-1beta in VSMCs could be attributed to 1) differential expression of ERalpha and ERbeta, 2) activation of distinct regulatory elements in the promoter of the iNOS gene as determined by the association of specific co-activator proteins with respective ER isoforms and 3) interaction between the liganded ER and the MAPK cascade activated by IL-1beta. The proposed studies will be conducted in VSMCs cultured from the mesenteric and coronary arteries of male and female rats. Identifying sites of estrogen's action in the regulation of gene expression in VSM could provide insight into the mechanisms by which estrogen exerts its cardioprotective effects in both sexes.